Measuring leaf area with ImageJ

Roman M. Link

Introduction

This is a simple tutorial for the measurement of leaf area with ImageJ based on this great blog post which is meant for in-class use.

Step-by-step tutorial

1. Open the calibration file with ImageJ (drag and drop onto the program or use the Open dialog).

2. Select the Straight line tool and follow the outer margin of the largest square in the calibration file.

3. Set the scale by going to the Analyze ➜ Set Scale menu.

4. In the corresponding dialog, set Known Distance to 4 cm, the Unit of length to cm and - very important - mark the box Global to make sure that the scale is the same accross all opened documents.

5. Open the file with the image you want to analyze in ImageJ (drag and drop!). If setting the scale worked, the size of the image should now be specified in cm in the upper right corner of the image. If a popup opens asking you whether you want to keep the global calibration, say yes!

6. Make sure that the image is in an 8bit black and white format (click on Image ➜ Type ➜ 8bit) to avoid problems with thresholding.

7. Set a black/white threshold

   • Open the Threshold dialog (Image ➜ Threshold or Ctrl + Shift + T),
   • Choose the options Default and B&W and uncheck the box Dark background,
   • If necessary, move the sliders until the leafs are well separated from the background with as few white pixels within the leaves as possible,
   • press Apply and close the Threshold window.

8. If there are holes (especially close to the leaf margins), pick the Pipette tool and click on a black area in the image.

9. Now, choose the Pencil tool and close the holes manually.

10. Open the set measurements dialog (Analyze ➜ Set Measurements), uncheck everything besides Area, and click OK.

11. Open the Analyze Particles dialog (Analyze ➜ Analyze Particles) and choose the following settings:

• Size: 0-Infinity
• Roundness: 0-1
• Show: Outlines
• Check Display results, Clear results and Include holes

12. Look at the outlines to check if non-leaf particles were measured.

• Solutions if there are small non-leaf particles:
  • Use the pencil tool to remove small artefacts
  • Use the rectangle tool (left end of the toolbar) to choose a subset of the Image area (useful if there are shading artefacts near the borders)
  • Repeat the Analyze Particles step with a minimum value for Size

initial run:

after setting Size to 1-Infinity cm²:

13. Click on the outline file and choose File ➜ Save as ➜ Jpeg in the main window to save it (append the original filename to name_outlines.jpg)

14. Choose File ➜ Save as in the Results window to save the leaf area measurements as name_results.xls.

15. Your project folder should look like this now: